Hydrogels are hydrophilic polymeric networks which can absorb and retain large amounts of water (Hoffman, 2002; Peppas, 1987; Peppas, 2006; Okano, 1998). Hydrogels are useful in controlled release systems for drug delivery (Kumar, 2002), tissue repair, tissue engineering (Cushing, 2007; Lee, 2001) and as surgical sealants and adhesives (Jackson, 1996; Spotnitz, 2001). Although great progress in medical applications of hydrogels has been made, it remains challenging to develop cross-linking methods that satisfy the demanding biological and handling requirements for medical treatment (Hennink, 2002). Accordingly, there is a long-felt, unmet need for biocompatible hydrogels capable of deployment by minimally invasive methods and solidification under physiological conditions.
Native chemical ligation (NCL) is a widely used technique for constructing a large polypeptide from two or more unprotected peptides. In NCL, a peptide containing a C-terminal thioester reacts with another peptide containing an N-terminal cysteine in the presence of an added thiol catalyst. In a freely reversible first step, a transthioesterification occurs to yield a thioester-linked intermediate; this intermediate rearranges irreversibly under the usual reaction conditions to form a native amide bond at the ligation site. Thioesters, for example, have proven useful in the chemical synthesis of large peptides and proteins using NCL (Dawson, 1994; Tam, 2000; Dawson, 2000; Macmillan, 2006; Paramonov, 2005). In nature, thioesters participate in the synthesis of a number of cellular components, and can be prepared as activated building blocks through chemical synthesis (Tam, 2000; Jakubowski, 1995; Kemp, 1981). Although relatively unreactive to aminolysis, thioesters readily react with a thiol group through transesterification to form a new thioester (Weber, 1979; Wieland, 1974; Wieland, 1953). The reaction between a thioester and an N-terminal-Cys yields an S-acyl covalent intermediate that spontaneously undergoes an S- to N-acyl migration to form an amide bond through a five-member ring intermediate (FIG. 15).
However, NCL has not been shown to successfully form hydrogels from soluble macromolecular precursors. NCL offers several advantages as a potential hydrogel cross-linking method, including chemoselectivity and high efficiency under physiological conditions without involving catalysts, initiators or other potentially toxic compounds. Further, NCL provides synthetic access to large peptides and proteins otherwise impossible to make, due to length or decoration by posttranslational modification. Further still, NCL cross-linking increases the stiffness of pre-assembled peptide hydrogels (Jung, 2008).
Accordingly, there is a long-felt, unmet need for biocompatible hydrogels and methods of synthesis and use thereof, where the hydrogels are synthesized using NCL.